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1.
Mitochondria complex II (succinate dehydrogenase, SDH) plays a central role in respiratory metabolism as a component of both the electron transport chain and the tricarboxylic acid cycle. We report the identification of an SDH assembly factor by analysis of T‐DNA insertions in At5g51040, a protein with unknown function that was identified by mass spectrometry analysis as a low abundance mitochondrial protein. This gene is co‐expressed with a number of genes encoding mitochondrial proteins, including SDH1‐1, and has low partial sequence similarity to human SDHAF2, a protein required for flavin‐adenine dinucleotide (FAD) insertion into SDH. In contrast to observations of other SDH deficient lines in Arabidopsis, the sdhaf2 line did not affect photosynthetic rate or stomatal conductance, but instead showed inhibition of primary root elongation with early lateral root emergence, presumably due to the low SDH activity caused by the reduced abundance of SDHAF2. Both roots and leaves showed succinate accumulation but different responses in the abundance of other organic acids and amino acids assayed. Isolated mitochondria showed lowered SDH1 protein abundance, lowered maximal SDH activity and less protein‐bound flavin‐adenine dinucleotide (FAD) at the molecular mass of SDH1 in the gel separation. The short root phenotype and SDH function of sdhaf2 was fully complemented by transformation with SDHAF2. Application of the SDH inhibitor, malonate, phenocopied the sdhaf2 root architecture in WT. Whole root respiratory assays showed no difference between WT and sdhaf2, but micro‐respirometry of the tips of roots clearly showed low oxygen consumption in sdhaf2 which could explain a metabolic deficit responsible for root tip growth.  相似文献   

2.
The pyruvate dehydrogenase complex (PDC), required for complete glucose oxidation, is essential for brain development. Although PDC deficiency is associated with a severe clinical syndrome, little is known about its effects on either substrate oxidation or synthesis of key metabolites such as glutamate and glutamine. Computational simulations of brain metabolism indicated that a 25% reduction in flux through PDC and a corresponding increase in flux from an alternative source of acetyl-CoA would substantially alter the 13C NMR spectrum obtained from brain tissue. Therefore, we evaluated metabolism of [1,6-13C2]glucose (oxidized by both neurons and glia) and [1,2-13C2]acetate (an energy source that bypasses PDC) in the cerebral cortex of adult mice mildly and selectively deficient in brain PDC activity, a viable model that recapitulates the human disorder. Intravenous infusions were performed in conscious mice and extracts of brain tissue were studied by 13C NMR. We hypothesized that mice deficient in PDC must increase the proportion of energy derived from acetate metabolism in the brain. Unexpectedly, the distribution of 13C in glutamate and glutamine, a measure of the relative flux of acetate and glucose into the citric acid cycle, was not altered. The 13C labeling pattern in glutamate differed significantly from glutamine, indicating preferential oxidation of [1,2-13C]acetate relative to [1,6-13C]glucose by a readily discernible metabolic domain of the brain of both normal and mutant mice, presumably glia. These findings illustrate that metabolic compartmentation is preserved in the PDC-deficient cerebral cortex, probably reflecting intact neuron–glia metabolic interactions, and that a reduction in brain PDC activity sufficient to induce cerebral dysgenesis during development does not appreciably disrupt energy metabolism in the mature brain.  相似文献   

3.
The mechanism of transduction of the phytochrome signal regulating the expression of succinate dehydrogenase in Arabidopsis has been investigated. Using the phytochrome mutants of Arabidopsis, it is demonstrated that the inhibition of succinate dehydrogenase in the light may result from the phytochrome A-dependent modulation of Ca2+ amount in the nuclear fraction of leaves. This leads to the activation of expression of the gene pif3 encoding the phytochrome-interacting factor PIF3, which binds to the promoter of the gene sdh1-2 encoding the SDHA subunit of succinate dehydrogenase and suppresses its expression. It is concluded that Ca2+ ions are involved in the phytochrome A-mediated inhibition of succinate dehydrogenase activity in the light.  相似文献   

4.
The activity of the respiratory enzyme fumarate reductase (FRD) is dependent on the covalent attachment of the redox cofactor flavin adenine dinucleotide (FAD). We demonstrate that the FAD assembly factor SdhE, which flavinylates and activates the respiratory enzyme succinate dehydrogenase (SDH), is also required for the complete activation and flavinylation of FRD. SdhE interacted with, and flavinylated, the flavoprotein subunit FrdA, whilst mutations in a conserved RGxxE motif impaired the complete flavinylation and activation of FRD. These results are of widespread relevance because SDH and FRD play an important role in cellular energetics and are required for virulence in many important bacterial pathogens.  相似文献   

5.
Recent studies have indicated that post-translational flavinylation of succinate dehydrogenase subunit A (SdhA) in eukaryotes and bacteria require the chaperone-like proteins Sdh5 and SdhE, respectively. How does covalent flavinylation occur in prokaryotes, which lack SdhE homologs? In this study, I showed that covalent flavinylation in two hyperthermophilic bacteria/archaea lacking SdhE, Thermus thermophilus and Sulfolobus tokodaii, requires heat and dicarboxylic acid. These thermophilic bacteria/archaea inhabit hot environments and are said to be genetically far removed from mesophilic bacteria which possess SdhE. Since mesophilic bacteria have been effective at covalent bonding in temperate environments, they may have caused the evolution of SdhE.  相似文献   

6.
Sabine Krawczyk 《FEBS letters》2010,584(8):1463-1020
In Corynebacterium glutamicum, the unphosphorylated 15-kDa OdhI protein inhibits the activity of the 2-oxoglutarate dehydrogenase complex (ODHc) by binding to OdhA, which in corynebacteria and mycobacteria is a large fusion protein with two major domains exhibiting structural features of E1o and E2 proteins. Using copurification and surface plasmon resonance experiments with different OdhI and OdhA length variants it was shown that the entire forkhead-associated (FHA) domain of OdhI and the C-terminal dehydrogenase domain of OdhA are required for interaction. The FHA domain was also sufficient for inhibition of ODHc activity. Phosphorylated OdhI was binding-incompetent and did not inhibit ODHc activity.

Structured summary

MINT-7713362:OdhI (uniprotkb:Q8NQJ3) binds (MI:0407) to OdhA (uniprotkb:Q8NRC3) by surface plasmon resonance (MI:0107)MINT-7713261:OdhI (uniprotkb:Q8NQJ3) physically interacts (MI:0915) with OdhA (uniprotkb:Q8NRC3) by pull down (MI:0096)  相似文献   

7.
8.
Kayode S. Oyedotun  Bernard D. Lemire 《BBA》2007,1767(12):1436-1445
The coupling of succinate oxidation to the reduction of ubiquinone by succinate dehydrogenase (SDH) constitutes a pivotal reaction in the aerobic generation of energy. In Saccharomyces cerevisiae, SDH is a tetramer composed of a catalytic dimer comprising a flavoprotein subunit, Sdh1p and an iron-sulfur protein, Sdh2p and a heme b-containing membrane-anchoring dimer comprising the Sdh3p and Sdh4p subunits. In order to investigate the role of heme in SDH catalysis, we constructed an S. cerevisiae strain expressing a mutant enzyme lacking the two heme axial ligands, Sdh3p His-106 and Sdh4p Cys-78. The mutant enzyme was characterized for growth on a non-fermentable carbon source, for enzyme assembly, for succinate-dependent quinone reduction and for its heme b content. Replacement of both Sdh3p His-106 and Sdh4p Cys-78 with alanine residues leads to an undetectable level of cytochrome b562. Although enzyme assembly is slightly impaired, the apocytochrome SDH retains a significant ability to reduce quinone. The enzyme has a reduced affinity for quinone and its catalytic efficiency is reduced by an order of magnitude. To better understand the effects of the mutations, we employed atomistic molecular dynamic simulations to investigate the enzyme's structure and stability in the absence of heme. Our results strongly suggest that heme is not required for electron transport from succinate to quinone nor is it necessary for assembly of the S. cerevisiae SDH.  相似文献   

9.
Comley John C. W. and Wright Spdenis J. 1981. Succinate dehydrogenase and fumarate reductase activity in Aspiculuris tetraptera and Ascaris suum and the effect of the anthelmintics cambendazole, thiabendazole, and levamisole. International Journal for Parasitology11: 79–84. Succinate dehydrogenase and fumarate reductase activities from a particulate fraction of A. tetraptera and a soluble extract of A. suum have been determined using spectrophotometric methods. Fumarate reductase activity in A. suum could only be detected anaerobically. Succinate dehydrogenase activity from A. suum was partially characterized and shown to exist in several multimolecular forms (isoenzymes). The in vitro effect of the anthelmintics cambendazole, thiabendazole and levamisole on succinate dehydrogenase and fumarate reductase activity from the above nematodes are described. Significant inhibition of fumarate reductase activity of both nematodes was only achieved using 5 mM levamisole and 1 mM thiabendazole. After in vivo anthelmintic treatment of A. tetraptera only thiabendazole significantly inhibited fumarate reductase. It is suggested that the succinate dehydro-ogenase-fumarate reductase complex in these nematodes is unlikely to be the primary site chemotherapeutic attack for any of the anthelmintics tested.  相似文献   

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13.
The functional state of the Photosystem (PS) II complex in Arabidopsis psbR T-DNA insertion mutant was studied. The ΔPsbR thylakoids showed about 34% less oxygen evolution than WT, which correlates with the amounts of PSII estimated from YDox radical EPR signal. The increased time constant of the slow phase of flash fluorescence (FF)-relaxation and upshift in the peak position of the main TL-bands, both in the presence and in the absence of DCMU, confirmed that the S2QA and S2QB charge recombinations were stabilized in ΔPsbR thylakoids. Furthermore, the higher amount of dark oxidized Cyt-b559 and the increased proportion of fluorescence, which did not decay during the 100s time span of the measurement thus indicating higher amount of YD+QA recombination, pointed to the donor side modifications in ΔPsbR. EPR measurements revealed that S1-to-S2-transition and S2-state multiline signal were not affected by mutation. The fast phase of the FF-relaxation in the absence of DCMU was significantly slowed down with concomitant decrease in the relative amplitude of this phase, indicating a modification in QA to QB electron transfer in ΔPsbR thylakoids. It is concluded that the lack of the PsbR protein modifies both the donor and the acceptor side of the PSII complex.  相似文献   

14.
Veena Prabhakar 《FEBS letters》2009,583(6):983-991
The Arabidopsis thaliana gene At1g74030 codes for a putative plastid phosphoenolpyruvate (PEP) enolase (ENO1). The recombinant ENO1 protein exhibited enolase activity and its kinetic properties were determined. ENO1 is localized to plastids and expressed in most heterotrophic tissues including trichomes and non-root-hair cells, but not in the mesophyll of leaves. Two T-DNA insertion eno1 mutants exhibited distorted trichomes and reduced numbers of root hairs as the only visible phenotype. The essential role of ENO1 in PEP provision for anabolic processes within plastids, such as the shikimate pathway, is discussed with respect to plastid transporters, such as the PEP/phosphate translocator.  相似文献   

15.
16.
The constitutive expression of the bacterial carotene desaturase (CRTI) in Arabidopsis thaliana leads to increased susceptibility of leaves to light-induced damage. Changes in the photosynthetic electron transport chain rather than alterations of the carotenoid composition in the antenna were responsible for the increased photoinhibition. A much higher level of superoxide/hydrogen peroxide was generated in the light in thylakoid membranes from the CRTI expressing lines than in wild-type while the level of singlet oxygen generation remained unchanged. The increase in reactive oxygen species was related to the activity of plastid terminal oxidase (PTOX) since their generation was inhibited by the PTOX-inhibitor octyl gallate, and since the protein level of PTOX was increased in the CRTI-expressing lines. Furthermore, cyclic electron flow was suppressed in these lines. We propose that PTOX competes efficiently with cyclic electron flow for plastoquinol in the CRTI-expressing lines and that it plays a crucial role in the control of the reduction state of the plastoquinone pool.  相似文献   

17.
18.
The mitochondrial succinate dehydrogenase (SDH) is an iron-sulfur flavoenzyme linking the Krebs cycle and the mitochondrial respiratory chain. Mutations in the human SDHB, SDHC and SDHD genes are responsible for the development of paraganglioma and pheochromocytoma, tumors of the head and neck or the adrenal medulla, respectively. In recent years, SDH has become recognized as a source of reactive oxygen species, which may contribute to tumorigenesis. We have developed a Caenorhabditis elegans model to investigate the molecular and catalytic effects of mutations in the sdhb-1 gene, which encodes the SDH iron-sulfur subunit. We created mutations in Pro211; this residue is located near the site of ubiquinone reduction and is conserved in human SDHB (Pro197), where it is associated with tumorigenesis. Mutant phenotypes ranged from relatively benign to lethal and were characterized by hypersensitivity to oxidative stress, a shortened life span, impaired respiration and overproduction of superoxide. Our data suggest that the SDH ubiquinone-binding site can become a source of superoxide and that the pathological consequences of SDH mutations can be mitigated with antioxidants, such as ascorbate and N-acetyl-l-cysteine. Our work leads to a better understanding of the relationship between genotype and phenotype in respiratory chain mutations and of the mechanisms of aging and tumorigenesis.  相似文献   

19.
GlnK proteins belong to the PII superfamily of signal transduction proteins and are involved in the regulation of nitrogen metabolism. These proteins are normally encoded in an operon together with the structural gene for the ammonium transporter AmtB. Haloferax mediterranei possesses two genes encoding for GlnK, specifically, glnK1 and glnK2. The present study marks the first investigation of PII proteins in haloarchaea, and provides evidence for the direct interaction between glutamine synthetase and both GlnK1 and GlnK2. Complex formation between glutamine synthetase and the two GlnK proteins is demonstrated with pure recombinant protein samples using in vitro activity assays, gel filtration chromatography and western blotting. This protein–protein interaction increases glutamine synthetase activity in the presence of 2-oxoglutarate. Separate experiments that were carried out with GlnK1 and GlnK2 produced equivalent results.  相似文献   

20.
D. Zannoni  B.L. Marrs 《BBA》1981,637(1):96-106
Membranes from cells of Rhodopseudomonas capsulata grown anaerobically in the dark on glucose plus dimethyl sulfoxide differ from those obtained from photoheterotrophically grown cells in several ways: (a) there are qualitative and quantitative variations in the cytochrome composition; (b) electron-transport rates are unusually low in the cytochrome b to cytochrome c region; (c) light-induced ATP synthesis is dependent on the ability of the alternate respiratory pathway to maintain the Q10-cytochrome b complex in a partially oxidized state; (d) a non-energy-conserving NADH-dehydrogenase activity dominates the respiratory activity. In addition, data obtained with both wild-type and mutant cells that contain altered electron-transport systems tend to exclude a role of the redox chain as ATP-producing machinery during anaerobic/dark growth.  相似文献   

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